Superoxide Detection Probes

Superoxide (O₂^-･) is a type of reactive oxygen species (ROS) generated by the one-electron reduction of molecular oxygen. It is also referred to as the superoxide anion, superoxide radical, or superoxide anion radical. Superoxide produced in biological systems can damage biomolecules because of its high reactivity. Prolonged exposure to excessive levels of reactive oxygen species--so-called oxidative stress--can lead to the development of various diseases, such as metabolic disorders and neurodegenerative diseases. Normal cells possess mechanisms to rapidly eliminate superoxide, collectively referred to as antioxidant capacity, and molecules involved in superoxide removal include nitric oxide (NO^･) and superoxide dismutase (SOD).

Superoxide is inherently difficult to measure directly because it is rapidly converted to hydrogen peroxide (H₂O₂) in polar, aqueous environments. Accurate detection therefore requires chemical reactions that proceed more rapidly than its conversion to hydrogen peroxide. Established methods include assays based on the reduction of cytochrome c or nitroblue tetrazolium (NBT)¹⁾. Fluorescent probes also offer a convenient approach for superoxide detection, and hydroethidine (HE) has been widely used for this purpose. However, hydroethidine has limited specificity for superoxide²⁾, underscoring the need for the development of fluorescent probes with improved specificity.

BES-So-AM and BES-So are fluorescent probes for detecting superoxide in living cells^2-3). These probes emit fluorescence through a redox-independent mechanism. Compared with the conventional probe, hydroethidine, they exhibit higher specificity for superoxide, resulting in reduced background signals. Therefore, they are suitable for live-cell imaging by fluorescence microscopy as well as for cell analysis by flow cytometry.

Features

• Detects superoxide in living cells
• Demonstrates higher specificity for superoxide than conventional fluorescent probes (hydroethidine)
• Water-soluble, allowing direct use in aqueous media
• Ex/Em=505 nm/544 nm

Principle

Data

Detection of Superoxide in Jurkat T Cells with BES-So-AM

Jurkat T cells were incubated in medium containing 33 μM BES-So-AM at 37°C for 1 hour to allow cellular uptake of the reagent. Superoxide production was induced by the addition of 5 mM butyrate, followed by an additional 1-hour incubation. In the control group, Tiron, a superoxide scavenger, was added to the medium together with BES-So-AM.

	Without O2-･ production / Without scavenger	With O2-･ production / Without scavenger	With O2-･ production / With scavenger
Fluorescence image
Phase-contrast image

Data by courtesy of Dr.Maeda, School of Pharmacy, Hyogo University of Health Sciences

1. 「活性酸素実験プロトコール」 ed. by Suma, H., Shujunsha, Tokyo, p.18(1994). (Japanese)
2. Maeda, H. et al.: J. Am. Chem. Soc., 127, 68(2005).
   A design of fluorescent probes for superoxide based on a nonredox mechanism
3. Maeda, H. et al.: Chem. Eur. J., 13(7), 1946 (2007).
   Design of a practical fluorescent probe for superoxide based on protection–deprotection chemistry of fluoresceins with benzenesulfonyl protecting groups.